Intravital multiphoton microscopy – Principles and challenges
Ken Dunn, PhD Scientific Director Indiana Center for Biological Microscopy Indiana University Medical Center In vivo imaging of the kidney
PET Ceccarini et al., 2009 Cell Metab.
Bioluminescence Que, Kajizel and Löwik, LUMC, Netherlands
MRI Bruker Biospin Intravital microscopy of the kidney
Wide-field microscopy Steinhausen et al., 1963.
Wide field microscopy Edwards and Marshall, 1924 Fluorescence microscopy Tanner et al., 1997, JASN Optical access to tissues in vivo
Oslo University Hospital Ritsma et al., Nature Protocols 2013
Dunn, Sutton and Sandoval. 2007. Curr. Protocols Cytometry. Holtmaat et al. Nature Protocols, 2009 Intravital microscopy of the kidney
Wide-field microscopy Steinhausen et al., 1963.
Wide field microscopy Edwards and Marshall, 1924
Dunn, Sandoval and Molitoris. 2003. Intravital imaging of the kidney using multiparameter multiphoton microscopy Nephron. 94:e7-11 Multiphoton fluorescence excitation
Fluorescence can be stimulated by the absorption of one photon of a particular energy level.
Maria Goppert-Mayer, 1931 – Fluorescence can also be stimulated by the simultaneous absorption of multiple, low-energy photons
Biophotonics Imaging Lab, Univ. Illinois Urbana-Champaign Two-photon action cross sections
10 kD Rhodamine Dextran, 500 kD Fluorescein Dextrn Hoechst 33342
Developmental Resource for Biophysical Imaging Opto-electronics, Cornell (Watt Webb and Warren Zipfel) Dunn et al., 2002. AJP Cell. 283:C905-C916 Relation between one and two photon absorption Not always 2X
Drobizhev et al., 2011. Xu et al., 1996. Bioimaging Nature Methods Multiphoton fluorescence excitation Requires an enormous density of photons
“Simultaneous absorption of two photons” – within ~ 10 attoseconds (10-17 seconds)
Detectible two-photon fluorescence excitation requires peak power on the order of 200 GW/cm2 ~ 300,000x the surface of the sun
Two photon fluorescence excitation first demonstrated with development of the ruby laser – Kaiser and Garrett, 1961 Biophotonics Imaging Lab, Univ. Illinois Urbana-Champaign Multiphoton fluorescence excitation Requires an enormous density of photons
Detectible two-photon fluorescence excitation requires peak power on the order of 200 GW/cm2 ~ 300,000x the surface of the sun
Two photon fluorescence excitation first demonstrated with development of the ruby laser – Kaiser and Garrett, 1961
For multiphoton microscopy, this density is provided by focusing laser illumination through high NA objective lenses. DiracDelta Science Photon density decreases with the 4th power of distance from the lens focus Multiphoton fluorescence excitation Requires an enormous density of photons
Detectible two-photon fluorescence excitation requires peak power on the order of 200 GW/cm2 ~ 300,000x the surface of the sun
Two photon fluorescence excitation first demonstrated with development of the ruby laser – Kaiser and Garrett, 1961
For multiphoton microscopy, this density is provided by focusing laser illumination through high NA objective lenses. Photon density decreases with the 4th power of distance from the lens focus Two-photon absorption occurs ONLY in the sub-femtoliter volume at the focus Multiphoton excited fluorescence is mapped to form an image
Build an image from single points
• A 2D image is formed by raster scanning a laser over the sample • A 3D image is formed by collecting a set of 2D images at different depths
Göbel and Helmchen 2007 Multiphoton excited fluorescence is mapped to form an image
Build an image from single points
• A 2D image is formed by raster scanning a laser over the sample • A 3D image is formed by collecting a set of 2D images at different depths • Image is formed by collecting, rather than imaging photons
Warren Zipfel Optimizing Microscope Design for Zipfel et al., 2003 Multiphoton Excitation Studies Wednesday at 9:20 am But 300,000 times brighter than the surface of the sun????
Denk, Strickler and Webb, 1990 - Use a pulsed laser ~100 femtosecond pulses at a rate of 80 MHz
Peak power sufficient for multiphoton excitation, but average power low enough to minimize damage.
Laser is mostly off – - Emitting only 0.001% of the time - Duty cycle equivalent to 1 sec pulse/day
Note that pulses do not REALLY limit the Dave Piston volume of excitation - Pulses ~ 45µ long and ~ 4 meters apart
Multiphoton microscope designs
Gregor Lab, Princeton Leica SP8 MP
Roberto Weigert Ken Dunn Building your own 2P microscope system Writing a Shared Instrumentation Grant Thursday, 9:20 am Thursday, 11:10 am
Fun facts about multiphoton microscopy
How frequently does multiphoton fluorescence excitation occur outside of a microscope?
Calculations made by Winfried Denk indicate that a molecule of rhodamine B exposed to direct sunlight will experience: • A one-photon absorption around once per second. • A two photon absorption once every 10,000 years. • A three-photon absorption . . . never in the history of the universe. Fun facts about multiphoton microscopy
Ridiculous time scales
Computer One Camera Human existence clock cycle month Age of flash 100 fs light pyramids pulse 1 minute Age of universe
-14 -9 -4 1 6 11 16 10 10 10 10 10 10 10 Time (seconds)
100 fs is to 10 minutes as 10 minutes is to the age of the universe.
Rick Trebino, University of Georgia Fun facts about multiphoton microscopy Relative time scales
Pixel Dwell Pulse Fluorescence time duration lifetime Two photon absorption 1 second
-18 -15 -12 -9 -6 -3 0 10 10 10 10 10 10 10 Time (seconds)
• Fluorescence lifetimes are ~ 1 billion times the length of the virtual intermediate state • At 80 MHz, the laser pulses are spaced ~ 3 fluorescence lifetimes apart (serendipitous) • A fluorophore will be illuminated ~320 times during a 4 microsecond pixel dwell time Why multiphoton microscopy?
Complex Expensive Poorer resolution Why multiphoton microscopy?
• IR light penetrates deeper, with less damage • no emission aperture - less loss to scattering Konig, 1999 • no photobleaching in out-of-focus planes
Srinivasan et al, 2003 Why multiphoton microscopy?
• IR light penetrates deeper, with less damage • Photobleaching only in the focal plane (but more of it)
Nikon MicroscopyU Why multiphoton microscopy?
Denk, 1996
• IR light penetrates deeper, with less damage • Photobleaching only in the focal plane (but more of it) • Optical sectioning without an emission aperture - less loss to scattering
Why multiphoton microscopy?
• IR light penetrates deeper, with less damage • Photobleaching only in the focal plane (but more of it) • Optical sectioning without an emission aperture - less loss to scattering
Centonze and White, 1998 Why multi-photon microscopy? Intravital microscopy
• Intravital microscopy provides submicron resolution and nanomolar sensitivity in under a second.
• Multiphoton microscopy has extended the reach of intravital microscopy to the scale of tissues and the functional components of organs. Applications of intravital multiphoton microscopy in biomedical research
Brain function and pathology • Neural development and activity - Winfried Denk, Karel Svoboda • Alzheimer's disease - Brian Bacskai, Brad Hyman • Astrocyte-neuron signaling - Jan Nedergard • Vascular function in the brain - David Kleinfeld Tumor biology • Tumor cell dynamics and metastasis - John Condelis, John Segal • Angiogenesis/vascular function, gene expression - Rakesh Jain Immunology • T-cell interactions and dynamics- Michael Dustin, Ullrich Von Andrian, Michael Cahalan • Immune surveillance in the brain - Fritjof Helmchen, W.B. Gan Liver function • Mitochondria and liver injury - John Lemasters • In vivo analysis of hepatobiliary transport – Ken Dunn Kidney function • Pathology and treatment of renal ischemia - Bruce Molitoris • Pathobiology of AKI - Katherine Kelly • Renal septic injury - Pierre Dagher Roberto Weigert • Microvascular function in renal injury - Tim Sutton Membrane Remodeling Through • Glomerular function - Janos Peti-Peterdi Membrane Trafficking in Live Animals Tuesday, 5pm
Dynamic intravital microscopy of the kidney
Ruben Sandoval and Bruce Molitoris Challenges of subcellular imaging in living animals
• Motion artifacts from respiration and heartbeat •Introduction of fluorescent indicators Reducing motion artifacts in intravital microscopy - Immobilize the organ
75 images collected over 30 min Reducing motion artifacts in intravital microscopy – Digital image registration
Before
Before After After
Presson, R., Brown, M., Sandoval, R., Dunn, K., Lorenz, K., Delp, E., Salama, P., Molitoris, B. and Petrache, I. 2011. Two-photon imaging within the murine thorax without respiratory and cardiac motion artifact. Am. J. Path. 179:75-82
Lorenz, K.S., Salama, P., Dunn, K.W. and E.J. Delp. 2011. Non-rigid registration of multiphoton microscopy images using B-splines. Progress in Biomedical Imaging SPIE proceedings. 7962. Reducing motion artifacts in intravital microscopy – Digital image registration
Dunn, Lorenz, Salama, and Delp. 2014. IMART software for correction of motion artifacts in images collected in intravital microscopy. Intravital. 3:e28210-1-10
Edward Delp Use of Digital Image Analysis for Studies of Renal Physiology Tuesday at 9:20 am Challenges of subcellular imaging in living animals
• Motion artifacts from respiration and heartbeat • Slow image capture rate Introduction of fluorescent indicators Single point scanning with a galvanometer 512 lines per second
Four 512x128 frames per second
One 512x512 frame per second
100 fps - widefield epifluorescence microscopy 1 minute, playing at 1/3rd speed Single point scanning with a resonant scanner 15000 lines per second (30 fps)
Fan et al., 1999. Biophys. Journal 76:2412 Speed limits for multi-photon microscopy
30-fold higher frame rates require 30-fold shorter pixel dwell times which then requires 30 fold better signal. For most applications increasing the signal requires increasing the excitation.
• In many applications of multiphoton microscopy, power A – 1 fps is already limiting – you may not be able to extract more signal with more power - 30 fold increase in signal requires 5.5 fold more power
• In many applications, multiphoton imaging is done at B – 28 fps power levels very close to fluorophore saturation – you may not get proportional increases in signal
• Photobleaching increases at greater than the cube of
Images of renal capillary of a living rat collected at power – disproportionately more photobleaching than 1 frame per second (A) or 28 fps (B). signal with more power
But, for experimental systems with sufficient fluorescence, resonant scanners are capable of capturing dynamics in vivo Try the Leica SP8 MP system Multi-point scanning multiphoton microscopy
• Multi-focal systems speed image acquisition by parallel scanning
• Power is divided among multiple excitation points – laser power limits multiplexing
• Imaging onto a CCD results in scattered emissions being attributed to the wrong position in the image, increasing background and reducing resolution
• The system would thus be of limited utility for imaging deep into tissues, the conditions that justify multiphoton microscopy in the first place Andresen et al., 2001. Optics Letters 26:75 Challenges of subcellular imaging in living animals
• Motion artifacts from respiration and heartbeat • Slow image capture rate • Limited reach/signal attenuation with depth • Introduction of fluorescent indicators Multiphoton microscopy Attenuation of signal with depth in the kidney
Glomeruli of most rats, mice are around 400 microns from the surface of the kidney
Cortico-medullary boundary is around 2 mm from the surface of the kidney
With a 60X NA 1.2 water immersion objective, signal attenuation prevents imaging deeper than around 100 microns in kidney of living rats Multiphoton microscopy Sources of signal attenuation of signal with depth
Dave Piston, Vanderbilt
• Light absorption • Light scattering • Spherical aberration Multiphoton microscopy Light absorption as a source of signal attenuation with depth
-az Model light extinction as - Iz = I0e Where
Iz = intensity of light at depth z a = the absorption coefficient For fluorescence excitation Near infrared light, a = .05 to 2 cm-1 in biological tissues So transmission attenuated by 50% in 0.35 - 14 cm But excitation is proportional to I2, Excitation is attenuated by 75% in 0.35 - 14 cm - Excitation reduced 75% in ~ 3500 to 140,000 microns - not a big deal
For collection of fluorescence emissions For 550 nm light, a = 4 to 20 cm-1 in biological tissues - Transmission reduced 50% in 350-1730 microns – not a real big deal
Most investigators agree, absorption is seldom a significant issue in biological tissues, except in skin (melanin) and under blood vessels (hemoglobin) Most investigators agree, absorption is seldom significant in biological tissues, except in skin (melanin) and under blood vessels (hemoglobin) Multiphoton microscopy Light scattering as a source of signal attenuation with depth
-sz Model light extinction as - Iz = I0e Where
Iz = intensity of light at depth z s = the scattering coefficient
For fluorescence excitation Near infrared light, s = 5 - 100 cm-1 in biological tissues So transmission attenuated by 50% in 70 to 1400 microns But excitation is proportional to I2, Excitation attenuated by 75% in 70 to 1400 microns
In general scattering is regarded as the predominant factor limiting depth of imaging in multiphoton microscopy Multiphoton microscopy Light scattering as a source of signal attenuation with depth
-sz Model light extinction as - Iz = I0e Where
Iz = intensity of light at depth z s = the scattering coefficient
For fluorescence excitation Near infrared light, s = 5 - 100 cm-1 in biological tissues So transmission attenuated by 50% in 70 to 1400 microns But excitation is proportional to I2, Excitation attenuated by 75% in 70 to 1400 microns For collection of fluorescence emissions For 550 nm light, s = 100 – 500 cm-1 in biological tissues So transmission attenuated by 50% in 15 - 70 microns
In general scattering is regarded as the predominant factor limiting depth of imaging in multiphoton microscopy Multiphoton microscopy Spherical aberration as a source of signal attenuation
Two photon microscopy of fluorescent beads mounted in different media using an oil-immersion objective
An oil-immersion objective imaging into an aqueous medium Surface 100um 100um 75um Aqueous Aqueous Aqueous 1.5 RI medium (w/out stretch)
Since the average refractive index of the kidney is ~1.4, we incur spherical aberration with either water immersion or oil immersion objectives Limited optical reach in the living kidney Potential solutions
• Reduce optical aberrations
• Jack up the laser Young, Clendenon, Byars, Decca and Dunn. 2011. J. Microscopy
• Longer wavelengths of light 22 61 microns microns • Improve fluorescence collection Olympus 25X, NA 1.05 Water immersion objective • Invade the kidney - "cut down",RI 1.33 GRIN lens, micro-prisms
19 67 • Reduce scattering – give up IVM microns microns Olympus 30X, NA 1.05 Silicone oil immersion objective RI 1.4 Limited optical reach in the living kidney Potential solutions
Adaptive optics
• Reduce optical aberrations
• Jack up the laser
• Longer wavelengths of light
Confocal microscope images of 1 micron fluorescent spheres located below 100 microns of kidney tissue before (A) and after • Improve fluorescence collection (B) adaptive optics correction – Joel Kubby, UCSC
• Invade the kidney - "cut down", GRIN lens, micro-prisms
• Reduce scattering – give up IVM
Seth Winfree and Ruben Sandoval Limited optical reach in the living kidney Potential solutions
• Reduce optical aberrations
• Jack up the laser
• Longer wavelengths of light
• Improve fluorescence collection
• Invade the kidney - "cut down",Imaging GRIN depth is ultimately limited by excitation of near-surface fluorescence lens, micro-prisms - signal-to-background ratio goes to 1 - Theer and Denk, 2006 • Reduce scattering – give up IVM Limited optical reach in the living kidney Potential solutions
Benefit of long-wavelength excitation
• Reduce optical aberrations
Tissue spheroid • Jack up the laser
• Longer wavelengths of light
Tumor xenograft
Andresen et al., 2009. Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging. Curr. Opin. Biotech. Limited optical reach in the living kidney Potential solutions
Benefit of long-wavelength emissions
• Reduce optical aberrations
• Jack up the laser
• Longer wavelengths of light Deliolanis et al., 2008. J. Biomed. Opt.
Drobizhev et al., 2011. Nature Methods Limited optical reach in the living kidney Three-photon fluorescence excitation
Benefits of 3-photon excitation at 1700 nm
• Reduce optical aberrations
• Jack up the laser
• Longer wavelengths of light
Mie scattering and water absorption as a function of wavelength
Signal-to-background ratio for 2P and 3P excitation
Horton et al., 2013. Nature Photonics Additional benefits of three-photon excitation
Third harmonic imaging Deep-UV excitation
Molecular Expressions
Deep tissue imaging of conventional Weigelin et al., 2016. J. Cell Science probes
Tim Harris Lab, Janelia Farms Limited optical reach in the living kidney Potential solutions
• Reduce optical aberrations
• Jack up the laser
• Longer wavelengths of light
• Invade the kidney - "cut down", GRIN lens, micro-prisms
Kidney “cut-down” or “parenchymal window” Limited optical reach in the living kidney Potential solutions
• Reduce optical aberrations
• Jack up the laser
• Longer wavelengths of light
• Invade the kidney - "cut down", GRIN lens, micro-prisms
Gradient Index (GRIN) lens Levene et al., 2004, J. Neurophysiol Limited optical reach in the living kidney Potential solutions
• Reduce optical aberrations
• Jack up the laser
• Longer wavelengths of light
• Invade the kidney - "cut down", GRIN lens, micro-prisms
Conventional XY image in XZ image in vivo with prism brain slice
Microprisms Chia and Levene. 2009. Limited optical reach in the living kidney Potential solutions
• Reduce optical aberrations
• Jack up the laser
• Longer wavelengths of light
• Invade the kidney - "cut down", GRIN lens, micro-prisms
Microprisms Gosia Kamocka and George Rhodes Limited optical reach in the living kidney Potential solutions
• Reduce optical aberrations
• Jack up the laser
Forbes et al., 2012. Fight-or-flight: murine unilateral ureteral • Longer wavelengths of light obstruction causes extensive proximal tubular degeneration, collecting duct dilatation and minimal fibrosis. AJP Renal 303 F120-129 • Invade the kidney - "cut down", GRIN lens, micro-prisms
• Bring the structures to the surface
Peti-Peterdi et al., 2015 Challenges of subcellular imaging in living animals
• Motion artifacts from respiration and heartbeat • Slow image capture rate • Limited reach/signal attenuation with depth • In vivo labeling •fluorescent indicators Sources of contrast in intravital multiphoton microscopy
• Autofluorescence
Hato, Ashkar and Dagher, 2013. AJP Renal
Takashi Hato Applications of micropuncture techniques and fluorescence lifetime imaging for intravital studies Wednesday at 12:10 am
Hall and Molitoris, 2014. AJP
Hato et al., 2017, JASN Sources of contrast in intravital multiphoton microscopy
• Autofluorescence • Hoechst 33342 Sources of contrast in intravital multiphoton microscopy
• Autofluorescence • Hoechst 33342 • Intravenous bulk probes Intravital microscopy of multiple kidney functions via intravenous injection of fluorescent probes
Simultaneous imaging of a rat injected with fluorescent dextrans and Hoechst 33342 reveals multiple processes
• glomerular filtration • proximal tubule endocytosis • tubular solute concentration • tubular flow • capillary blood flow • vascular permeability • apoptosis • tubular sloughing
Dunn et al., 2002. AJP Cell. 283:C905-C916 Sources of contrast in intravital multiphoton microscopy
Rhodamine 123 in liver mitochondria – John Lemasters • Autofluorescence • Hoechst 33342 • Intravenous bulk probes • Permeant bio-indicators
DCFDA labeling S2 cells in oxidative stress Pierre Dagher
Pierre Dagher Fluorescent probes for studying AKI Tuesday at 11:10 am Kalakeche et al., 2011. JASN
Tim Sutton Using fluorescent probes to study CMFDA delivered to cytosol by subcapsular injection tubule metabolism Rudy Juliano, UNC Thursday at 8:30 am
In vivo labeling of renal macrophages
Calcein loaded into macrophages by phagocytosis of pH-sensitive liposomes containing calcein Takashi Hato and Pierre Dagher (30x)
Calcein-loaded pH-sensitive liposomes provided by Rudy Juliano, UNC Sources of contrast in intravital multiphoton microscopy
• Autofluorescence • Hoechst 33342 • Intravenous bulk probes • Permeant bio-indicators
• Second harmonic imaging Fibrotic mouse kidney, Vuillemin et al., 2016. Scientific Reports
Liver of living mouse Gosia Kamocka and Amy Zollman
Ex vivo human sweat gland Mary Beth Brown Sources of contrast in intravital multiphoton microscopy
Fluorescence, SHG and THG excitation at 1180 nm • Autofluorescence • Hoechst 33342 • Intravenous bulk probes • Permeant bio-indicators • Second harmonic imaging • Third harmonic imaging
Weigelin et al., 2016. J. Cell Science Sources of contrast in intravital multiphoton microscopy
• Autofluorescence
Prostate tumor • Hoechst 33342 Tom Gardner • Intravenous bulk probes • Permeant bio-indicators • Second harmonic imaging • Third harmonic imaging • Adoptive transfer
Pancreatic islets transplanted into mouse kidney Jennifer Ryan and Natalie Stull Sources of contrast in intravital multiphoton microscopy
• Autofluorescence
• Hoechst 33342 GFP-expressing hematopoetic stem cells in mouse calvarium • Intravenous bulk probes Gosia Kamocka, Amy Zollman, Nadia Carlesso • Permeant bio-indicators • Second harmonic imaging • Third harmonic imaging • Adoptive transfer • Transgenic animals
Fluorescent neutrophils in liver of GFP-Lys mouse Cliff Babbey, Marwan Ghabril and Ken Dunn Sources of contrast in intravital multiphoton microscopy
• Autofluorescence • Hoechst 33342 • Intravenous bulk probes • Permeant bio-indicators
• Second harmonic imaging Micropuncture injection of Adeno-GFP-actin Ashworth et al., 2007 • Third harmonic imaging • Adoptive transfer • Transgenic animals • In vivo gene transfer
Hydrodynamic delivery ofAdeno-GFP-actin Corridon et al., 2013 Sources of contrast in intravital multiphoton microscopy
Intravenous injection of virus • Autofluorescence • Hoechst 33342 • Intravenous bulk probes
• Permeant bio-indicators Mouse liver Adeno-AKAR4.1 • Second harmonic imaging • Third harmonic imaging • Adoptive transfer • Transgenic animals • In vivo gene transfer
Mouse pancreas – AAV8-GFP Mouse kidney intravenous AAV9-GFP Sources of contrast in intravital multiphoton microscopy
Subcapsular injection of virus • Autofluorescence • Hoechst 33342 • Intravenous bulk probes • Permeant bio-indicators • Second harmonic imaging • Third harmonic imaging • Adoptive transfer
• Transgenic animals Adeno-RGD-GFP AAV9--GFP • In vivo gene transfer
Bob Bacallao Expanding the toolbox for intravital imaging of the kidney using gene delivery techniques Wednesday at 8:30 AM
Challenges of subcellular imaging in living animals
• Motion artifacts from respiration and heartbeat • Slow image capture rate • Limited reach/signal attenuation with depth • In vivo labeling • Extracting quantitative data despite depth attenuation fluorescent indicators Quantitative intravital microscopy circumventing the effects of depth on signal levels
• Quantify without intensity –Structural changes –Motion Amyloid plaque dynamics - Brian Bacskai, Harvard –Incidence
• Quantify to reference intensity –Ratiometric measures –Time-series measures
Dendritic spine dynamics - Karel Svoboda, Janelia Farm Quantitative intravital microscopy circumventing the effects of depth on signal levels
• Quantify without intensity –Structural changes –Motion Sham LPS LPS + APC –Incidence
• Quantify to reference intensity –Ratiometric measures –Time-series measures
Microvascular flow
Sharfuddin Sandoval, Berg, McDougal, Campos, Phillips, Jones, Gupta, Grinnell and Molitoris. 2009. Soluble thrombomodulin protects ischemic kidneys. J Am Soc Nephrol:524-34. Quantitative intravital microscopy circumventing the effects of depth on signal levels
• Quantify without intensity –Structural changes –Motion –Incidence
• Quantify to reference intensity Neutrophil migration – Paul Kubes, Calgary –Ratiometric measures –Time-series measures
Quantitative intravital microscopy circumventing the effects of depth on signal levels
• Quantify without intensity –Structural changes –Motion –Incidence
–Incidence
• Quantify to reference intensity –Ratiometric measures –Time-series measures
Neutrophil migration – Paul Kubes, Calgary Quantitative intravital microscopy circumventing the effects of depth on signal levels
• Quantify without intensity –Structural changes –Motion –Incidence
• Quantify relative intensities –Ratiometric measures
Richard Day Fluorescent proteins and biosensors Tuesday at 10:20 pm PKA activation in mouse liver– AKAR4.1 FRET sensor Practical method for monitoring FRET-base biosensor probe Forster Resonance Energy Transfer and activities using two-photon excitation microscopy Tao et al., in prep Fluorescence Lifetime Microscopy Wednesday at 11:10 am Quantitative intravital microscopy circumventing the effects of depth on signal levels
• Quantify without intensity –Structural changes –Motion –Incidence
• Quantify relative intensities –Ratiometric measures
Microvascular leakage in the bone marrow space of the mouse calvarium
Malgorzata Kamocka, Amy Zollman and Nadia Carlesso - IUSM Quantitative intravital microscopy circumventing the effects of depth on signal levels
• Quantify without intensity –Structural changes –Motion –Incidence Vehicle treated Rifampin treated Effect of rifampin on fluorescein transport • Quantify relative intensities
2500 –Ratiometric measures
2000 Control canal –Time-series measures Control cytosol Rifampin cytosol 1500
1000
Mean fluorescence (AU) 500
0 0 1 2 3 4 5 6 7 Minutes after injection
Babbey et al., 2012. Quantitative intravital microscopy of hepatic transport. Intravital. 1:44-53 Further information
Websites
http://www.drbio.cornell.edu/ - Watt Webb’s laboratory http://www.microscopyu.com/ - a great general microscopy education website http://www.loci.wisc.edu - Laboratory for Optical and Computational Instrumentation
Reviews
Girkin, J. 2003. Optical physics enables advances in multiphoton imaging. J. Phys. D: Appl. Phys. 36:R250-R258.
Helmchen, F. and W. Denk. 2005. Deep tissue two-photon microscopy. Nature Methods 2:932-940
Hoover, E. and J. Squier. 2013. Advances in multiphoton microscopy technology. Nature Photonics. 7:93-101
Zipfel, W., R. Williams and W. Webb. 2003. Nonlinear magic: multiphoton microscopy in the biosciences. Nature Biotech. 21:1369-1377.
The Indiana OBrien Center for Advanced Renal Microscopy